An anti-hepatitis B virus surface antigen antibody (anti-HBsAg antibody) is produced after infection of hepatitis B virus or vaccination of hepatitis B virus vaccine, and used to monitor results of hepatitis B virus vaccine by measuring a concentration of the produced anti-HBs antibody through anti-HBs antibody titer test (anti-HBs concentration of 10 mIU/mL or less is determined as a non-immune anti-HBs concentration). In addition, in a liver transplant having hepatitis B by hepatitis B virus as an underlying disease, the anti-HBsAg antibody is used to monitor a titer of the anti-HBs antibody at the time of administrating hepatitis B immunoglobulin (HBIG) in order to prevent reinfection of the hepatitis B virus.
As a method for analyzing a titer of the anti-HBs antibody, there are an enzyme immunoassay (EIA), chemiluminescent microparticle immunoassay (CMIA), radioimmunoassay (RIA), and the like. (El-Madhun et al., Vaccine, 16:156-160, 1998; L. Haaheim et al., International Congress Series, 1219:283-289, 2001; Odd Odinsen et al., Clin. Vaccine Immunol., 14(10):1623-1628, 2007; P. Kryger et al., J. Clin. Microbiol., 13:405-409, 1981).
The method for analyzing the titer of the anti-HBs antibody is to measure the titer of the antibody by coating a surface antigen (HBsAg) of the hepatitis B virus on a microwell plate or microparticles, reacting the surface antigen with a sample to be measured, and then measuring an amount of color development, luminescence, or isotopes by using the surface antigen (HBsAg) of the hepatitis B virus conjugated with an enzyme or a radioactive isotope.
IgG which is a representative antibody has a Y shape. As illustrated in FIG. 1A, antigen binding sites are present at both ends of the Y shape, such that the antigens are bound to each site. As illustrated FIG. 1A, measuring the titer is normally performed when a surface antigen of the hepatitis B virus coated on the bottom (hereinafter, referred to as a coated HBsAg) is bound to one site of the antigen binding sites of the anti-HBs antibody, and a surface antigen of the hepatitis B virus labeled with an enzyme or a radioactive isotope (hereinafter, referred to as a labeled HBsAg) is bound to the other site, that is, when the surface antigens are bound to each site.
However, when measuring a titer of the anti-HBs monoclonal antibody developed by Green Cross Corp., according to a method illustrated in FIG. 1A without human plasma or serum, the measured titer is about 20 to 100 times lower than that of a method illustrated in FIG. 1D. It is thought that the reason that the anti-HBs titer of the anti-HBs monoclonal antibody measured by the method of FIG. 1A is lower may actually come from an error possibility due to a combination such as FIG. 1B or FIG. 1C. That is, a case in which two binding sites of the antibody to be measured are bound to both of the coated HBsAg or the labeled HBsAg (FIG. 1B), or a case in which one binding site of the antibody is not capable of being bound to one antigen when the other binding site of the antibody is bound to the antigen (FIG. 1C) may occur, and accordingly, an amount of the antibody may not be accurately measured.
In addition, even though the titer is measured by the method illustrated in FIG. 1D to overcome the above-described problems, when a fully human antibody such as the anti-HBs monoclonal antibody developed by Green Cross Corp., is used, non-specific binding of the fully human antibody with other human antibodies originally present in human plasma or serum may occur as illustrated in FIG. 2, and a secondary antibody recognizing a protein comprising human Fc is not capable of differentiating the non-specific binding and a specific binding of the anti-HBs monoclonal antibody, which causes high background noise, and accordingly, it is still difficult to normally measure the titer.
In order to solve the above-described disadvantages, the present inventors found that measurement error may be reduced by accurately measuring only specific reactions such as FIG. 1D through the method for measuring a titer based on indirect ELISA; however, precision in the measurement of the antibody titer among test subjects is still low.
Accordingly, the present inventors made an effort to exclude error possibility of the existing titer measurement and increase precision in the titer measurement of the a protein comprising human Fc in the human plasma or serum, and as a result, found that when a kit for measuring a titer of a protein comprising human Fc including a diluent for sample, a diluent for conjugate, and a cleansing solution each having a specific composition, is used, the error possibility in measuring the titer of the a protein comprising human Fc in the human plasma or serum is remarkably decreased, and the precision in the measurement of the antibody titer among test subjects is increased, and completed the present invention.